Walton_unc_0153D_15258.pdf

THE CONTRIBUTION OF FIBRINOGEN AND RED BLOOD CELLS TO ARTERIAL THROMBOSIS

Bethany Lynne Walton

“A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department

of Pathology and Laboratory Medicine in the School of Medicine.”

Chapel Hill 2015

Approved by: Alisa S. Wolberg William B. Coleman

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© 2015

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ABSTRACT

Bethany Lynne Walton: The Contribution of Fibrinogen and Red Blood Cells to Arterial Thrombosis

(Under the direction of Alisa S. Wolberg)

Cardiovascular disease is the leading cause of death and disability worldwide. This dissertation explores the role of the clotting factor fibrinogen and red blood cells (RBCs) to arterial thrombosis.

Elevated plasma fibrinogen is associated with arterial thrombosis in humans and directly promotes thrombosis in mice, but the contribution of the γA/γ′ fibrinogen isoform to thrombosis is controversial. To determine if γA/γ′ is prothrombotic, we separated γA/γA and γA/γ′ from human plasma and determined the effects on in vitro clot formation and on in vivo

thrombus formation. Both γA/γA and γA/γ′ were cleaved by murine and human thrombin and were incorporated into murine and human clots. When γA/γA or γA/γ′ was spiked into plasma, γA/γA increased the fibrin formation rate to a greater extent than γA/γ′. In mice, compared to controls, γA/γA infusion shortened the time to carotid artery occlusion, whereas γA/γ′ infusion did not. Additionally, γA/γ′ infusion led to lower levels of plasma thrombin–

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ACKNOWLEDGEMENTS

I would like to express my gratitude for the Wolberg Laboratory for serving as an excellent place to perform this dissertation work. Alisa has been a wonderful mentor to me and has helped guide me through every step of this research over the past 5 years. Thank you to both current and former lab members for keeping the lab a fun place to work everyday. I have also been fortunate to have the support of faculty, administration, and graduate students within the Pathology Department. Thanks to the members within the UNC Hemostasis and Thrombosis group and our many collaborators for their scientific input and experimental help. Thank you to my committee members, Drs. Alisa Wolberg, Bill Coleman, Brian Cooley, Nigel Key, and Leslie Parise, for their expertise to guide my research. Also, thank you to my funding sources including the UNC Program in Translational Research and the Integrative Vascular Biology program for helping provide the funds needed to complete this dissertation.

Thank you to all my supportive and caring friends in Chapel Hill for filling my life outside the lab with so many great memories. A special thanks to all the pub runners for hours upon hours of running and great conversation. Thanks to BAKTDL for being my best friends since elementary school. I would especially like to thank my amazing family

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TABLE OF CONTENTS

LIST OF TABLES……….x

LIST OF FIGURES………..xi

LIST OF ABBREVIATIONS AND SYMBOLS………...xiii

CHAPTER 1: INTRODUCTION: FIBRINOGEN AND RED BLOOD CELLS IN HEMOSTASIS AND THROMBOSIS………..…1

1.1 Introduction………..…1

1.2 Fibrinogen………....2

1.2.1 Fibrinogen structure, fibrin formation and fibrin mechanical properties………...…....2

1.2.2 Fibrinogen and arterial and venous thrombosis………...…….3

1.2.3 Abnormal fibrin structure and stability in thrombosis………..………....3

1.2.4 γ’ fibrinogen and arterial and venous thrombosis …………..………..…4

1.2.5 Fibrinogen interaction with cells and blood proteins………...….8

1.3 Red Blood Cells………...8

1.3.1 RBCs in circulation………...8

1.3.2 Causes of high hematocrit………...9

1.3.3 Hematocrit and arterial and venous thrombosis………9

1.3.4 RBCs mediate blood rheology………...……….13

1.3.5 RBC interaction with fibrinogen……… 13

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1.3.7 RBCs influence platelet reactivity………...………...16

1.3.8 RBCs and thrombin generation……….……..16

1.3.9 RBCs alter fibrin structure & stability………...………...19

1.4 Conclusions………....19

1.5 Focus of this Dissertation………...19

REFERENCES………....21

CHAPTER 2: THE FIBRINOGEN γA/γ’ ISOFROM DOES NOT PROMOTE ACUTE ARTERIAL THROMBOSIS IN MICE………32

2.1 Introduction………..……..32

2.2 Materials and Methods………...………34

2.2.1 Proteins and Materials……….…………34

2.2.2 Plasma Preparation……….……….35

2.2.3 Isolation of γA/γA and γA/γ’ fibrinogen...……….35

2.2.4 SDS-PAGE and western blotting………36

2.2.5 Clot formation with purified fibrin(ogen)………...36

2.2.6 Clot formation in plasma………36

2.2.7 Intravital microscopy………..36

2.2.8 FeCl3 thrombosis model……….…….37

2.2.9 Measurement of circulating TAT complexes……….38

2.2.10 Statistical Methods………38

2.3 Results………38

2.3.1 γA/γA fibrinogen increases the fibrin polymerization rate to a greater extent than γA/γ’ fibrinogen……….38

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into murine thrombi in vivo……….47

2.3.3 Following FeCl3 injury, γA/γA, but not γA/γ’, fibrinogen shortens the time to artery occlusion……….49

2.3.4 Following FeCl3 injury mice infused with γA/γ’ fibrinogen have lower circulating TAT complexes than mice infused with γA/γA fibrinogen………51

2.4 Discussion………..53

REFERENCES………..………..59

CHAPTER 3: ELEVATED HEMATOCRIT PROMOTES ARTERIAL THROMBOSIS INDEPENDENT OF THROMBIN GENERATION………64

3.1 Introduction……….……64

3.2 Materials and Methods………66

3.2.1 Proteins and materials……….…66

3.2.2 Phlebotomy……….67

3.2.3 Reconstituted whole blood model……….…..67

3.2.4 Thrombin generation in whole blood……….….…68

3.2.5 Mouse model of elevated hematocrit………..…68

3.2.6 Blood smears………...…69

3.2.7 Flow cytometry………...69

3.2.8 Blood viscosity measurements………....70

3.2.9 FeCl3 model of arterial thrombosis………...…..…70

3.2.10 Measurement of circulating thrombin-antithrombin (TAT) complexes ………..…71

3.2.11 Histology………...…71

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3.3 Results……….72

3.3.1 The RBC increase in thrombin generation in whole blood is dependent on platelet concentration………72

3.3.2 RBC infusion raises hematocrit in mice without altering RBC morphology or PS-exposure………..75

3.3.3 Compared to controls, RBCHIGH mice have a faster time to arterial occlusion………...77

3.3.4 In vivo thrombin generation did not differ between control and RBCHIGH mice……….79

3.3.5 Thrombi from control and RBCHIGH mice do not differ in size or fibrin content……….…..…81

3.3.6 PF4 levels did not differ between control and RBCHIGH mice…………83

3.4 Discussion………..…….85

REFERENCES………..………..90

CHAPTER 4: SUMMARY AND FUTURE DIRECTIONS………..…95

4.1 Summary………....95

4.2 Future Directions……….…..95

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LIST OF TABLES

Table 1.1 Epidemiological studies are conflicted on the association

between hematocrit and arterial thrombosis………....11 Table 1.2 Epidemiological studies are conflicted on the

association between hematocrit and venous thrombosis……….……12 Table 2.1 Polymerization of purified fibrinogen isoforms by human

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LIST OF FIGURES

Figure 1.1 The fibrinogen γ chain undergoes alternative processing to

form the γA and γ’ isoforms………..…7 Figure 1.2 RBCs marginate platelets toward the vessel wall under

arterial shear………...…………..14 Figure 1.3 A portion of RBCs express PS on their cell

membrane.………18 Figure 2.1 Purified fibrinogen contains all three chains

(Aα, Bβ, and γA and/or γ’) at the expected molecular weights

and is equally cleaved by human and mouse thrombin …………...……...…40 Figure 2.2 Both γA/γA and γA/γ’ fibrinogen accelerate clotting in human

and mouse plasma………...…44 Figure 2.3 Intravital microscopy shows both γA/γA and γA/γ’ isoforms

are incorporated into murine thrombi………..……48 Figure 2.4 γA/ γA fibrinogen shortens the time to vessel occlusion after

arterial injury, but γA/γ’ does not………50 Figure 2.5 Following arterial injury, mice infused with γA/γ’ fibrinogen

have reduced circulating TAT complexes………...52 Figure 2.6 γA/γ’ fibrinogen binds thrombin, resulting in lower

thrombin-antithrombin levels and similar TTO as controls ………...55 Figure 3.1 The RBC effect on thrombin generation

is dependent on platelet concentration………...74 Figure 3.2 RBC transfusion raises hematocrit in recipient mice………...76 Figure 3.3 RBCHIGH mice have a shortened time to vessel occlusion following

FeCl3 injury to the carotid artery, ………..78 Figure 3.4 Thrombin-antithrombin complexes are similar in RBCHIGH mice

and controls following FeCl3 injury……….………80

Figure 3.5 Thrombi from control and RBCHIGH mice have similar

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Figure 4.1 Conceptual model showing influence of RBCs on thrombus

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LIST OF ABBREVIATIONS AND SYMBOLS

α Alpha

AU Arbitrary Units

β Beta

BSA Bovine Serum Albumin °C Degrees Celsius

CaCl2 Calcium Chloride

CAT Calibrated Automated Thrombography CGS Sodium citrate, Glucose, Sodium Chloride CHD Coronary Heart Disease

CVD Cardiovascular disease DVT Deep Vein Thrombosis

ELISA Enzyme-Linked Immunosorbent Assay FeCl3 Ferric Chloride

FXIII Factor XIII

γ Gamma

γ’ Gamma Prime

g Gram

H&E Hematoxylin and Eosin HBS HEPES Buffered Saline

hNPP Human Normal Pooled Plasma

Hz Hertz

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ICAM Intercellular Adhesion Molecule IVC Inferior Vena Cava

K Thousand

KCl Potassium Chloride

L Liters

µL Microliter µM Micromolar

M Molar

mg Milligram

MgSO4 Magnesium Sulfate Min Minute

MI Myocardial infarction mL Milliliter

mM Millimolar

mNPP Murine Normal Pooled Plasma NaCl Sodium Chloride

nm Nanometer

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PRP Platelet-rich Plasma PS Phosphatidylserine PV Polycythemia Vera RBCs Red Blood Cells

RBCHIGH Mice with Elevated Hematocrit SCD Sickle Cell Disease

SD Standard Deviation SE Standard Error

SEM Standard Error of the Mean TAT Thrombin-antithrombin TF Tissue Factor

tPA Tissue-type Plasminogen Activator TTO Time to Occlusion

VTE Venous Thromboembolism WBC White Blood Cell

Chapter 1: Introduction: Fibrinogen and Red Blood Cells in Hemostasis and Thrombosis1

1.1 Introduction

Arterial thrombosis is a leading cause of death and disability worldwide. Arterial

thrombosis is usually initiated following rupture of an atherosclerotic plaque. This causes the

formation of thrombi that may become occlusive and cause ischemic damage to the

surrounding tissues. Intracardiac thrombosis may also occur due to atrial fibrillation or the

presence of a mechanical valve [1]. Arterial thrombi are usually termed “white-thrombi” due

to their high platelet count and efforts to understand the pathogenesis of arterial thrombosis

have mainly focused on platelets. However growing evidence suggests that the plasma

protein fibrin(ogen) and RBCs may also be involved in the development of arterial thrombi.

Venous thrombosis is initiated by endothelial dysfunction and inappropriate

expression of plasma and cellular procoagulant activity under low blood flow/stasis

(so-called Virchow’s Triad). The epidemiology, risk factors, and treatment of venous thrombosis

have been recently reviewed in [2]. However, the pathophysiologic mechanisms that

contribute to thrombus formation, composition, and stability are still poorly understood.

Clues may be found in the distinctive appearance of venous thrombi, which demonstrate

regions of high RBC and fibrin content (so-called “red thrombi”). Notably, RBCs can be

found between layers of fibrin in a “brick and mortar” construction, where they lose their

typical discoid shape and acquire a compressed morphology (so-called “polyhedrocytes”) [3].

These observations suggest RBCs and fibrin(ogen) interact during venous thrombosis, and

that thrombi undergo substantial consolidation during their maturation.

Herein, I will review the contributions of fibrinogen and RBCs to coagulation, and

provide evidence supporting their potential roles in both arterial and venous thrombosis.

1.2 Fibrinogen

1.2.1 Fibrinogen structure, fibrin formation, and fibrin mechanical properties.

The fibrinogen molecule consists of 2 sets each of 3 polypeptide chains (AαBβγ)2.

During coagulation, thrombin cleaves N-terminal peptides from the Aα- and Bβ-chains

promoting the formation of protofibrils and subsequently, fibrin fibers. Branching results in

the characteristic fibrin network seen in micrographs of purified and plasma clots.

Fibrinogen circulates at high concentrations (2-4 mg/mL) in plasma, and levels may increase

further during inflammation. The concentrations of thrombin and fibrinogen present during

clot formation influence fibrin network structure and stability. For example, clots formed in

the presence of high thrombin or fibrinogen concentrations have increased fibrin network

density and resistance to fibrinolysis compared to clots formed under normal conditions.

These processes have been previously reviewed [4, 5].

Crosslinked fibrin is also known for its ability to stabilize clots. This property is

determined at both micro- and macro-scales. Individual fibrin fibers have astounding

viscoelasticity. Crosslinked fibrin fibers can be stretched to 2.5-times their original length

of fibers from elongations up to 100% can occur within milliseconds [9]. Branchpoints within the fibrin network are surprisingly strong. When strained, individual fibers fail before branchpoints fail [10]. Thus, it is not surprising that fully-formed fibrin clots have similar extensibility and elasticity as individual fibers.

1.2.2 Fibrinogen and arterial and venous thrombosis.

Elevated total fibrinogen is correlated with increased arterial thrombosis [11-15] and increased venous thrombosis risk [16-20], and risk is concentration-dependent and present in both men and women. Studies using transgenic mice and murine infusion models have associated elevated fibrinogen with increased prothrombotic biomarkers (e.g., D-dimer) [21] and a shorter time to vessel occlusion and increased thrombus fibrin content [22]. Moreover, compared to control mice, thrombi in fibrinogen-infused mice also show increased resistance to fibrinolysis [22]. These findings suggest hyperfibrinogenemia is not merely a biomarker of thrombosis risk, but is causative in arterial and venous thrombosis etiology.

1.2.3 Abnormal fibrin structure and stability in thrombosis.

Several studies have reported abnormal fibrin structure and/or stability in both arterial and venous thrombosis, even when circulating fibrinogen levels are normal. For example, compared to controls, patients with a history of MI produced clots with shorter fibrin fibers with increased stiffness, and an increased resistance to lysis [23, 24]. Additionally,

fibrin network density, reduced permeability, and increased lysis times[27]. Interestingly, compared to plasma clots from patients with deep vein thrombosis, clots from patients that experienced pulmonary embolism are less compact and more susceptible to fibrinolysis [27, 28]. In total, these data suggest abnormal fibrin network structure and stability contribute to arterial and venous thrombosis.

1.2.4 Fibrinogen γ’-chain and arterial and venous thrombosis.

The fibrinogen γ-chain can undergo alternative splicing, leading to replacement of 4 C-terminal amino acids with a unique 20 amino acid sequence (γ’; Figure 1.1). The γ’-chain is present in 8-15% of fibrinogen molecules (as γA/γ’) in healthy individuals. The genes encoding the fibrinogen chains are co-regulated to maintain the level of fibrinogen in

circulation (reviewed in [29]). However, the levels of the γA- and γ’-chains are mediated by independent mechanisms that differentially regulate their expression. Expression of γ’-containing fibrinogen is disproportionally increased by interleukin-6-dependent

inflammatory responses [30], suggesting an independent relationship between the γ’-chain, inflammation, and thrombosis. Accordingly, although total fibrinogen levels are positively correlated with thrombosis risk, the fraction of circulating γ’-fibrinogen (γ’/total fibrinogen ratio) modulates risk independently of the total fibrinogen level. Notably, an elevated γ’-to-total fibrinogen ratio is associated with increased risk of arterial thrombosis in numerous epidemiological studies [31-36], suggesting γ’ may be driving arterial thrombosis.

Determining the operant mechanisms has been difficult because γA/γ’ fibrinogen has

both procoagulant and antithrombotic properties (reviewed in [39]). Briefly, compared to

γA/γA clots, clots that contain γ’ fibrinogen have a denser network of thin fibrin fibers,

reduced permeability, reduced plasminogen binding, and increased resistance to fibrinolysis.

The γ’-chain can also bind and sequester thrombin, protecting it from inactivation by

antithrombin. These properties are consistent with prothrombotic functions. However, γ’

fibrinogen also exhibits impaired polymerization. Recent studies have shown that a γ’

carboxyl-terminal peptide reduces plasma thrombin generation even in the presence of

anti-factor VIII antibody, suggesting γ’/thrombin interactions reduce anti-factor V activation [40]. By

reducing thrombin generation, this peptide also increases the sensitivity of coagulation to

activated protein C, thus augmenting endogenous anticoagulant mechanisms [41].

Studies to determine the contribution of the γ’-chain to thrombosis in vivo have

consistently demonstrated antithrombotic effects. Transgenic expression of the human

γ’-chain reduces venous thrombus volume in mice that are heterozygous for the factor V Leiden

mutation [42]. A peptide mimicking the γ’-chain C-terminus inhibits fibrin-rich thrombus

formation in a baboon model of thrombosis [43]. We recently infused mice with identical

levels of either γA/γA or γA/γ’ fibrinogen isolated from human plasma [44]. Compared to

controls, γA/γA infusion shortens the time to carotid artery occlusion, whereas γA/γ’ infusion

does not. Additionally, γA/γ’ infusion reduces levels of circulating thrombin-antithrombin

complexes. These data are consistent with the premise that the γ’-chain reduces thrombin

activity. By extension, these data implicate the γA-chain as the prothrombotic mediator in

Figure 1.1: The fibrinogen γ chain undergoes alternative processing to form the γA and γ’ isoforms. The fibrinogen γ-chain mRNA transcript may undergo splicing at two main polyadenylation sites. The γA-chain forms when polyadenylation (pA) occurs downstream of exon 10 (pA2), leading to translation of exon 10 and the formation of a γ-chain with 10 exons, 9 introns, and 411 amino acids (ending in AGDV). The γ’-chain forms when

polyadenylation occurs upstream of exon 10 (pA1), forming a γ-chain which includes intron 9. This results in the translation of an extra 20 amino from intron 9

(VRPEHPAETRYDSLYPEDDL), forming a γ-chain with 427 amino acids.

EXON%9% INTRON%9% EXON%10%

pA2$

EXON%9% EXON%10% AGDV$

pA1$

Fibrinogen$Gamma$Chain$Gene$

γA$Fibrinogen$

pA2$

VRPEHPAETEYDSLYPEDDL$ EXON%9% INTRON%9%

pA1$

1.2.5 Fibrin(ogen) interactions with cells and blood proteins.

Most studies of fibrin(ogen) function have used purified systems or plasmas. These studies have identified binding sites on fibrin(ogen) for soluble proteins involved in clot formation, stabilization, and fibrinolysis, including thrombin, FXIII, fibronectin, tissue-type plasminogen activator (tPA), plasminogen, and plasmin [45-49]. Notably, however,

fibrin(ogen) also interacts with cells and these interactions may contribute to the

incorporation of cells into venous thrombi. For example, fibrin(ogen) contains recognition sequences for integrins including αMβ2, αIIbβ3, αVβ3, which mediate fibrin(ogen)

interactions with leukocytes, platelets, and endothelial cells, respectively [50]. These interactions modulate leukocyte function, platelet aggregation and clot retraction, and may anchor thrombi to the endothelium. Fibrin(ogen) also binds to RBCs, which influences both the erythrocyte sedimentation rate and blood viscosity (discussed below) [51-53].

1.3 Red Blood Cells

1.3.1 RBCs in circulation.

1.3.2 Causes of high hematocrit.

The normal range of RBCs in blood (hematocrit) is 41-46% in men and 36-44% in women, and can be influenced by a number of physiologic and pathologic situations. In high altitude, the bone marrow increases RBC production to compensate for decreased oxygen saturation [62]. RBC levels may also increase in disease states such as polycythemia vera (PV) [63] and as a result of increased erythropoietin, either through exogenous erythropoietin use or abnormal erythropoietin production by certain types of tumors [64]. Conditions associated with hypoxia, such as smoking, lung disease, and heart disease, are also associated with increased RBC production [64].

1.3.3 Hematocrit in arterial and venous thrombosis.

Thrombosis is a common complication in patients with PV, with arterial thrombosis including MI and cerebrovascular events making up the majority of all thrombotic events in these patients [65]. The Cytoreductive Therapy in Polycythemia Vera (CYTO-PV), a large-scale, multicenter, prospective, randomized clinical trial compared maintaining hematocrit <45% or between 45-50% in patients with PV [66]. Compared to maintaining a hematocrit <45%, maintaining hematocrit in the higher range was associated with four times the rate of death from CVD and major thrombosis suggesting hematocrit is a cause for thrombosis in PV. However, this study did not control for patients taking hydroxyurea which complicates the interpretation of these results.

Table 1.1 Epidemiological studies are conflicted on the association between hematocrit and arterial thrombosis

Study N Methods Results

Sorlie (1981) Puerto Rico Health Program [58] -2555 Rural/6151 urban men (45-64 years old)

-Subjects examined 3 times in 8 year follow-up

-High HCT (>49%)

associated with an increased risk of MI, coronary

insufficiency, and CHD death compared to low HCT (<42%).

-Risk more than double in high HCT group

Carter (1983) [68] -8006 Japanese men -Subjects completed questionnaires & underwent interviews & medical screening -10 year follow-up

-HCT correlated with predictors or CHD risk, related to non-fatal MI, CHD death, and total CHD

-When adjusted for other variables, HCT not independent risk factor Gagnon

(1994) Framingham Study [60]

-5209 men & women (30-62 years old) -1073 deaths

-Subjects received biannual examinations -34 year follow-up

-Men and women in the highest HCT quintile had increased risk of CVD death (men ≥49%; women ≥46%) -Correlation strongest in younger men Brown (2001) Second National Health & Nutrition Examination Survey (NHANES II) Mortality Study [67]

-8896 men & women (30-75 years old)

-HCT categorized by sex-specific tertiles -16.8 year follow-up

-Women in the upper tertile were 1.3 times more likely to die from CHD than women with HCT in lowest tertile after multivariate adjustment

-Risk was stronger in women <65 years old -HCT not associated with CVD or death in men after multivariate adjustment Kunnas (2009) TAMRISK Study [69] -670 Finnish Men (55 years old)

-188 deaths from CVD

-Health survey data taken on 670 men in 1980

-28 year follow-up -Divided into two groups-HCT ≥50 or <50

-Men with HCT >50% were 2.4 more likes to die from CHD than men with HCT <50%

-After adjusting for CHD risk factors, risk was 1.8 fold Toss (2013)

[61]

-417,099 Swedish men (18-19 years old) -9322 1st MI

-Baseline health tests and measures during conscription

-36 year follow-up

-HCT ≥49% had a 1.4 fold increased risk of MI

compared to men with HCT ≤44%.

Table 1.2 Epidemiological studies are conflicted on the association between hematocrit and venous thrombosis

Study N Methods Results

Tsai (2002) The Longitudinal Investigation of Thromboembolism Etiology [70]

19292 men & women

-Combines data from, the Atherosclerosis Risk In Communities (ARIC) study and Cardiovascular Heath Study (CHS)

-8 year follow-up

-No relation between HCT and VTE -Limitation: Low HCT cut-off at 43.5%

Vaya (2002) [71] -109 1st time VTE patients -121 controls

-Subjects had no inherited or acquired risk factors for DVT -DVT documented with ultrasonography or venography and PE with ventilation perfusion scanning or pulmonary

angiography

-Blood collected from patients 6-36 months after the VTE episode (85% collected within the first year)

-No relation between HCT, RBC

aggregation, plasma viscosity and DVT

Braekkan (2010) Tromsø Study [72]

-26108 men & women

- 12.5 year follow-up -Multivariate hazard ratios per 5% increase in HCT were 1.25 for total VTE and 1.37 for unprovoked VTE. -Men with HCT in upper 20th _percentile had a 1.5-fold

1.3.4 RBCs mediate blood rheology.

Hemorheology is the study of how flowing blood influences hemostasis. RBCs are the major determinant of blood rheology because of their prevalence, size, deformability, and ability to undergo reversible aggregation. Under high shear in the arterial circulation,

(typically 500-1500 s-1), RBCs promote platelet flux toward the vessel wall (so-called platelet margination), which increases the frequency of platelet-endothelial cell interactions, and platelet-platelet interactions which promotes platelet adhesion, activation, and

aggregation (Figure 1.2) [73]. However, under low shear in the venous circulation (typically 10-100 s-1), RBCs increase blood viscosity via their tendency to aggregate (rouleaux

formation). Increased blood viscosity is a risk factor for arterial and venous thrombosis [74-76]. Notably, RBC aggregation is mediated by plasma proteins including fibrinogen [51-53]. Consequently, inflammatory processes that increase fibrinogen levels also increase blood viscosity. These effects have been implicated in the association between elevated hematocrit and hyperfibrinogenemia with thrombosis. However, it remains unclear whether this

Figure 1.2: RBCs marginate platelets toward the vessel wall under arterial shear. RBCs dominate the rheology of blood due to their high number, large size, and deformability. Under the high shear rates present in arteries, RBCs move toward the center of the blood vessel. This marginates platelets towards the arterial walls, which promotes increased platelet-platelet interactions and platelet-endothelial interactions.

RBC$ Platelets$

Blood$Flow$

Smooth$Muscle$Cell$ Endothelial$Cell$

1.3.5 RBCs interact with fibrin(ogen).

RBCs interact specifically with fibrin(ogen) and the fibrinogen motif that mediates

RBC interactions involve fibrinogen Aα-chain residues 207-303 [51]. Two potential RBC

receptors have been implicated in this interaction. Fibrinogen-RBC interactions can be

inhibited by the integrin-blocking molecule eptifibatide and are not supported by RBCs

lacking β3 isolated from patients with Glanzmann thrombasthenia [77], implicating β3 or a

β3-like molecule on the RBC surface. However, that study did not rule out the possibility that RBC-bound platelets mediate this interaction [77]. Fibrinogen-RBC interactions can also be

blocked with an antibody against the integrin-associated protein CD47 [78]. Since CD47 was

originally identified for its interaction with αvβ3, αIIbβ3, and α2β1 integrins, it is possible

that the RBC binding site comprises a complex with both of these molecules. It is

interesting to speculate that that blocked fibrin(ogen)-RBC interactions may reduce whole

blood viscosity and thus thrombosis risk.

1.3.6 RBCs interact with cells.

RBCs can interact with leukocytes, platelets, and endothelial cells. For example,

RBC ICAM-4 can bind leukocyte β1 and β2 integrins [79, 80] and platelet αIIbβ3 [81]. RBC

ICAM-4 also interacts with integrin αv [82]. RBCs are the first cells to adhere to ferric

chloride (FeCl3)-treated, intact arterial endothelium, prior to the arrival of platelets [83]. This

interaction is not dependent on von Willebrand factor or GPIbα. However, the molecular

receptors on RBCs and the endothelium that mediate this interaction have not been identified

[84], suggesting stage-specific receptors may decorate RBCs during differentiation and

further refine these interactions.

1.3.7 RBCs influence platelet reactivity.

RBCs have been shown to alter the biochemical responsiveness and functional

responsiveness of activated platelets [85]. Silvain et al. [86] showed that RBCs increase

ADP-induced platelet activation and aggregation in vitro in blood from healthy volunteers.

The same group went on to show that patients who received RBC transfusions displayed

increased ADP-induced platelet reactivity [87]. Moreover, it was specifically shown that

RBCs amplify platelet activation and degranulation by increasing platelet serotonin release,

increasing enzymatic ADP removal, and inhibiting proteases [85]. The increase in platelet

reactivity by RBCs could not be decreased by aspirin, suggesting that RBC level influences

therapeutic effect of aspirin. RBCs have also been shown to enhance the activation of αIIbβ3

and P-selectin on platelets, suggesting RBCs increase platelet activation and aggregation [88].

In sum, these studies suggest that RBCs contribute to thrombus formation by increasing

platelet reactivity.

1.3.8 RBCs support thrombin generation.

A small percentage (~0.5%) of RBCs circulate with exposed phosphotidylserine (PS)

on their outer membranes [89], suggesting RBCs can assemble prothrombinase complexes

and support thrombin generation (Figure 1.3). Interestingly, although levels of both

PS-positive RBCs and platelets are elevated in patients with sickle cell disease (SCD) genotypes,

including F1.2 and D-dimer [89]. This finding suggests PS-positive RBCs are the primary cell responsible for thrombophilia in SCD. In vitro studies support this premise; when added to platelet-poor plasma, RBCs shorten the lag time and increase the peak of thrombin

generation similar to that seen with platelets [90, 91], although in contrast to platelets, thrombin generation on RBCs occurs through the meizothrombin pathway [92]. RBCs can

Figure 1.3: A portion of RBCs express PS on their cell membrane. A small percentage (0.5%) of RBCs express PS exposure on their cell surface. This surface allows for

prothrombinase complex assembly, prothrombin conversion to thrombin, and fibrin formation. Fibrin can then be crosslinked by the transglutaminase factor XIII, to form the structural support for a clot.

II

Prothrombin

IIa

Thrombin Xa# Va#

Prothrombinase

Fibrinogen

Fibrin Fibrin

Fibrin TF#

Tissue Factor

Fibrin Fibrin Fibrin RBC#

1.3.9 RBCs alter fibrin structure and stability.

RBCs alter fibrin network structure [96, 97] and reduce fibrin network permeability [98]. RBCs also suppress plasmin generation and reduce clot dissolution [97]. In the

presence of the substantial contractile forces induced by platelets during clot retraction [99, 100], RBCs are dramatically compressed, which further reduces clot permeability and

restricts access of fibrinolytic enzymes to the clot [3, 101]. Importantly, this phenomenon was noted in thrombi harvested from the arterial vasculature [3] but is also likely to have a significant impact on venous thrombosis, since these thrombi contain platelets and large

numbers of RBCs. These data suggest reducing RBC content in thrombi may increase clot dissolution, thus reducing thrombosis.

1.4 Conclusions

Both fibrinogen and RBCs are essential components of blood and major players in

coagulation. Continued studies are needed to delineate the pathophysiologic mechanisms that mediate the roles of both fibrinogen and RBCs in thrombosis. Specifically, identifying

the specific fibrinogen isoform that promotes thrombosis and knowledge of the role of elevated hematocrit in thrombosis will provide new insight into mechanisms that drive abnormal clot formation. Additionally, identification of the RBC-receptor that binds

fibrinogen may provide new therapeutic targets to prevent thrombosis.

1.5 Focus of this dissertation

total fibrinogen (combined γA/γA and γA/γ’) and previous work on RBCs in arterial

thrombosis has been performed in murine models with co-morbidities. Therefore, this

dissertation will specifically focus on i) the effect of the γA/γA and γA/γ’ individually to

arterial thrombosis, and ii) the influence of elevated hematocrit to arterial thrombosis in a

RBCs transfusion model of elevated hematocrit. Knowledge on how fibrinogen isoforms and

RBCs contribute to thrombosis may help elucidate mechanisms involved in the formation of

arterial thrombi, reveal biomarkers to predict thrombosis, and provide clues into the best

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Chapter 2: The fibrinogen γA/γ’ isoform does not promote acute arterial thrombosis in mice2

2.1 Introduction

Fibrinogen is a 340 kDa glycoprotein that circulates in plasma at 2-4 mg/mL, but

during acute inflammation can exceed 7 mg/mL. Fibrinogen is composed of two sets of three

polypeptide chains: Aα, Bβ, and γ. Alternative splicing of the main γA chain leads to the γ’

chain. Molecules containing the γ’ chain circulate as a heterodimer with the γA chain (2Aα,

2Bβ, and γA/ γ’) and comprise 8-15% of total fibrinogen in healthy individuals [1, 2].

Elevated fibrinogen levels are associated with increased risk of arterial thrombosis [3-5], and

we previously showed that when mice are infused with unfractionated human fibrinogen

(~90% γA/γA and 10% γA/γ’) and subjected to FeCl3-mediated carotid artery injury, elevated

plasma fibrinogen shortens the time to vessel occlusion [6]. These findings suggest elevated

fibrinogen is a causative, etiologic agent in arterial thrombosis. However, the specific

contributions of γA/γA and γA/γ’ fibrinogen isoforms to thrombosis in vivo are unknown.

In vitro studies to define the biochemical role of the γ’ chain have shown that clots made with purified γA/γ’ fibrinogen polymerize at a slower rate than clots made with

purified γA/γA fibrinogen [7]. Additionally, the γ’ chain supports high affinity binding to

thrombin exosite II [8, 9], and studies have shown that thrombin binding to the γ’ chain

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

2_{!This chapter is based on and reproduced in part with permission from:}

Walton BL, Getz TM, Bergmeier W, Lin FC, Uitte de Willige S, Wolberg AS.

competitively inhibits mediated platelet activation [10] and reduces

thrombin-mediated FpB cleavage [7], and factor VIII [11] and V [12] activation. These properties

suggest γA/γ’ fibrinogen has anticoagulant activity in vitro. Conversely, the γ’ chain does

not inhibit thrombin-mediated cleavage of FpA [7, 13], and has been reported to support

higher affinity binding of FXIII than the γA chain [14], although more recent studies suggest

only slightly tighter [14], or even similar [15], binding of FXIII to the γA/γ’ isoform

compared to the γA/γA isoform. Additional studies in purified systems report contradictory

effects of the γ’ chain on clot structure and mechanical properties, demonstrating that the γ’

chain induces the formation of alternately smaller [7, 13, 16] or larger [17] pores, and stiffer

[18] or less stiff [17] clots. These conflicting observations make it difficult to predict the role

of γA/γ’ fibrinogen under physiologic conditions in thrombosis in vivo.

The role of the human γ’ chain in thrombosis has previously been tested in two in

vivo studies. Since the murine γ’ chain does not contain the thrombin-binding sequence

found on the human γ’ chain, Mossesson et al. developed a transgenic mouse that replaced

the murine γ’ chain with the human γ’ chain [19]. Following electrolytic injury to the

femoral vein, there was no difference in thrombus volume between mice containing the

human γ’ chain and wild type (WT) controls, although the presence of the human γ’ chain

reduced thrombus volume in mice that were also heterozygous for the factor V Leiden

mutation [19]. However, interpretation of these findings is complicated by the higher total

fibrinogen in WT mice compared to mice expressing the human γ’ chain. In a baboon model

in which an arteriovenous shunt was placed between the femoral artery and vein, an 18

thrombus formation [11]. These studies suggest the γ’ chain reduces fibrin accumulation and

is antithrombotic during venous thrombosis.

Given these findings, it is interesting that retrospective epidemiological studies have

correlated elevated γA/γ’ fibrinogen levels with increased incidence of coronary artery

disease [20], myocardial infarction [21], and stroke [22-24]. In particular, the finding that

some patients have an increased γ’-to-total fibrinogen ratio [22-25] indicates γA/γ’

fibrinogen is not merely a biomarker of increased total fibrinogen, and suggests a specific

role for γA/γ’ in arterial thrombosis. However, these studies do not and cannot demonstrate

causality of γ’ chain-containing fibrinogen in thrombosis. The objective of our study was to

determine the contribution of γA/γA and γA/γ’ fibrinogen to arterial thrombosis.

2.2 Materials and Methods

2.2.1 Proteins and Materials.

Polyclonal rabbit anti-human fibrinogen antibody was from DAKOCytomation

(Carpinteria, CA). Monoclonal anti-fibrin(ogen) antibody (59D8) was a generous gift of Drs.

Marschall Runge (University of North Carolina), Charles Esmon (Oklahoma College of

Medicine), and Rodney Camire (University of Pennsylvania). Mouse anti-human γ’

chain-specific antibody (2.G2.H9) was from Millipore (Temecula, CA). Biotinylated secondary

antibodies were from Vector Laboratories (Burlingame, CA). The AlexaFluor-488 protein

labeling kit and 10% pre-cast Tris-glycine gels were from Invitrogen (Carlsbad, CA).

Human α-thrombin and murine thrombin were from Enzyme Research Laboratories (South

Bend, IN). Lipidated tissue factor (TF, Innovin) was from Siemens (Newark, DE).

were prepared as described [26]. Bovine serum albumin was from Sigma-Aldrich (St. Louis,

MO). Peroxidase substrate was from KPL (Gaithersburg, MD).

2.2.2 Plasma preparation.

Contact-inhibited human normal pooled plasma (hNPP) was prepared from 40

healthy subjects (50% female, 68% nonwhite) as described [27], in a protocol approved by

the UNC Institutional Review Board. γA/γ’ fibrinogen levels in hNPP were measured by

ELISA, as described [28]. Murine normal pooled plasma (mNPP) was prepared by

collecting blood from 49 female C57Bl/6 mice by inferior vena cava (IVC) venipuncture into

3.2% sodium citrate (1:9 ratio sodium citrate:blood). Pooled whole blood was centrifuged

(4000xg, 20 minutes), and platelet-poor plasma was aliquoted and frozen at -80oC.

2.2.3 Isolation of γA/γA and γA/γ’ fibrinogen.

The γA/γA and γA/γ’ fibrinogen variants were separated from human plasminogen-,

von Willebrand Factor-, and fibronectin-depleted human fibrinogen (Enzyme Research

Laboratories Ltd., Swansea, UK), based on the method described previously [7]. After

purification, variants were concentrated using Vivaspin 20 MWCO 100,000 columns (GE

Healthcare, Uppsala, Sweden) and dialyzed into 20 mM N-2-hydroxyethylpiperazine-N′

-2-ethanesulfonic acid (pH 7.4) containing 150 mM NaCl (HBS). Fibrinogen concentrationwas

determined by absorbance at 280 nm using an extinctioncoefficient of 1.51 mL/(mg/cm).

2.2.4 SDS-PAGE and western blotting.

Fibrinogen preparations were assessed by 10% SDS-PAGE and Coomassie Brilliant

Blue staining or western blotting for total fibrinogen or fibrinogen γ’ chain. For western

blots, membranes were blocked with Tris-buffered saline with 1% Tween containing 5%

milk, washed, and probed sequentially with mouse-anti human γ’-specific primary antibody

and AlexaFluor-488 conjugated anti-mouse secondary antibody. Fluorescent signal was

detected on a Typhoon 900 FLA fluorescent scanner.

2.2.5 Clot formation with purified fibrin(ogen).

Purified fibrinogen, thrombin, and CaCl2 (0.5 mg/mL, 5 nM, and 10 mM, final,

respectively) were combined in 96-half-well plates and polymerization was monitored by

turbidity at 405 nm using SpectraMax Plus340 plate reader (Molecular Devices, Sunnyvale,

CA).

2.2.6 Clot formation in plasma.

hNPP or mNPP was spiked with HBS (Control), or γA/γA or γA/γ’ fibrinogen, and

clotting was initiated with TF (1:30,000 dilution of Innovin, final), 10 mM CaCl2, and 4 µM

phospholipid vesicles in 96-well plates. Clot formation was monitored by turbidity at 405

nm.

2.2.7 Intravital microscopy.

Procedures were approved by the UNC Institutional Animal Care and Use Committee.

Briefly, 6-8 week old male C57Bl/6 mice (Charles River Laboratories, Wilmington, MA) were anesthetized and laser injuries were induced with an Ablate! photoablation system equipped with an attenuatable 532 nm pulse laser (Intelligent Imaging Innovations, Denver,

CO). Five minutes before injury, mice were injected via the retro-orbital plexus with AlexaFluor 595-labeled anti-glycoprotein IX antibody (0.3 mg/g body weight; Emfret,

Eibelstadt, Germany), and AlexaFluor 647-labeled murine anti-fibrin antibody (0.2 mg/g body weight), and trace amounts (5% of total fibrinogen) of AlexaFluor 488-labeled γA/γA or γA/γ’ fibrinogen. Five venules maximum were studied per mouse.

2.2.8 FeCl3 thrombosis model.

FeCl3 injury to carotid arteries was performed as described [6]. Briefly, 6-8 week old male C57Bl/6 mice were anesthetized, and human fibrinogen or vehicle (HBS) was

administered through the left saphenous vein cannula on a per-weight basis 5 minutes before

injury. The right common carotid artery was exposed, dried and treated with FeCl3 (10% on 0.5×1.0-mm filter paper) for 2 minutes. We specifically titrated the conditions to perform

these experiments at a threshold at which some mice do not form thrombi, to allow for sensitivity to both increased and decreased procoagulant activity. Blood flow was monitored by Doppler ultrasonic flow probe, and the time to occlusion (TTO) was defined as the time

2.2.9 Measurement of circulating TAT complexes.

TAT levels were measured by ELISA (Enzygnost TAT micro ELISA, Siemens) using

plasma prepared from IVC blood draws from mice subject to FeCl3 carotid artery thrombosis

(blood was drawn ~5 minutes following vessel occlusion). Samples showing hemolysis were

excluded.

2.2.10 Statistical Methods.

Descriptive statistics (mean, median, standard deviation [SD], standard error of the

mean [SEM]) were calculated. Groups were compared using Student’s t-tests

(normally-distributed data determined by Lilliefors test for normality) or Wilcoxon-Mann-Whitney

Rank Sum Tests (non-normally distributed data) in Kaleidagraph v4.1.3. Correlations were

performed using SAS 9.2 (SAS Inc., Cary, NC). P<0.05 was considered statistically

significant.

2.3 Results

2.3.1 γA/γA fibrinogen increases the fibrin polymerization rate to a greater extent than

γA/γ’ fibrinogen.

Purified γA/γA fibrinogen contained all three fibrinogen chains (Aα, Bβ, and γ) at

expected molecular weights (Figures 2.1A-B). No γ’ chain was detected in γA/γA fibrinogen

(Figure 2.1C), whereas purified γA/γ’ fibrinogen showed equal intensities of γA and γ’ bands

(Figures 2.1A-B). We first clotted purified fibrinogens with purified human thrombin and

followed clotting by turbidity. Although fibrinogen γA/γA and γA/γ’ isoforms were not

FXIII does not affect differences in polymerization between γA/γA and γA/γ’ fibrinogen [17]. Indeed, consistent with previous reports [7, 13, 17], purified γA/γA exhibited a faster

Table 2.1. Polymerization of purified fibrinogen isoforms by human and murine thrombin

Human Thrombin Murine Thrombin

Lagtime (seconds)

Change in Turbidity (OD)

Vmax

(mOD/min) Lagtime (seconds)

Change in Turbidity (OD)

To determine the effect of elevated γA/γA and γA/γ’ fibrinogen on plasma clot

formation during in situ thrombin generation, we spiked purified γA/γA, γA/γ’, or HBS

(control) into hNPP. The concentration of fibrinogen in hNPP was 3.1±0.1 mg/mL (100%)

and baseline concentration of γA/γ’ fibrinogen in hNPP was 0.42 mg/mL (13.5% of total

fibrinogen). We increased the total fibrinogen concentration to 3.5 (114%), 3.9 (127%), or

4.4 (143%) mg/mL by spiking in purified γA/γA or γA/γ’, so that the γA/γ’-to-total

fibrinogen ratios ranged from 9.6-40.1% (Table 2.2). These levels span the range of γA/γ’

levels measured in healthy individuals and patients with thrombosis [23-25, 30, 31].

Elevating either γA/γA or γA/γ’ fibrinogen increased final clot turbidity compared to plasma

spiked with HBS (Figure 2.2B, Table 2.2). When total fibrinogen was raised to 114%,

neither γA/γA nor γA/γ’ fibrinogen increased the clot formation rate. However, elevating

total fibrinogen to 127% or 143% with γA/γA or γA/γ’ significantly and dose-dependently

increased the clot formation rate versus baseline (HBS). Notably, at each concentration,

elevating total fibrinogen with γA/γA increased the clot formation rate to a significantly

greater extent than elevating total fibrinogen with γA/γ’ (Figure 2.2C, Table 2.2). Linear

regression analysis showed that the clot formation rate correlated positively with elevated

total fibrinogen (r=0.667, P<0.001) and negatively with the γ’-to-total fibrinogen ratio

(r=-0.0245, P=0.17), although the relationship between γ’-to-total and clot formation rate did not

reach significance. Moreover, the level of γA/γA isoform correlated strongly with the clot

formation rate (r=0.795, P<0.001) whereas the level of γA/γ’ did not.

Spiking purified human γA/γA, γA/γ’, or HBS (Control) into mNPP produced similar

results. For these experiments, the fibrinogen concentration in mNPP was 2.4±0.2 mg/mL

yielding human γ’-to total fibrinogen ratios ranging from 0-41.2%. Consistent with previous

observations [6], the final turbidity of murine plasma clots was lower than that of human

plasma clots, likely reflecting increased fibrin density of murine fibrin networks versus

human networks (unpublished observation). As in human plasma, both γA/γA and γA/γ'

increased the clot formation rate, but γA/γA increased the rate to a greater extent than γA/γ'

at each concentration tested (P<0.02, Figure 2.2F, Table 2.3). These findings suggest that

during in situ thrombin generation, both elevated γA/γA and γA/γ’ fibrinogen promote clot

Figure 2.2. Both γA/γA and γA/γ’ fibrinogen accelerate clotting in human and mouse plasma. A-C) hNPP was spiked with γA/γA or γA/γ’ to increase total fibrinogen to 114%, 127%, or 143% of normal (symbols appear in figure legend), and clot formation was

triggered by addition of TF and CaCl2. D-F) mNPP was spiked with human γA/γA or γA/γ’

to increase total fibrinogen to 135% or 170% of normal (symbols appear in figure legend) and clot formation was triggered by addition of TF and CaCl2. A, D) Polymerization was

monitored by turbidity; for clarity, only a subset of points is shown. B, C, E, F) The